Dry Season Ecology of Malaria Vectors Our work continues to focus on: (1) Windborne long-range migration of mosquitoes as strategies of vector and malaria persistence in the Sahel, and (2) Mosquito dormancy (aestivation) through the dry season to account for the persistence of A. coluzzii . Building on previous results, we have amassed additional compelling evidence that A. coluzzii (previously, the M form of A. gambiae) persists locally by aestivation whereas A. gambiae (previously, the S form) and A. arabiensis re-colonize the area after the onset of rains. To explain the long-distance migration our project combines (i) on-the-ground monitoring of vector density and composition along 9 surveillance villages, spanning over 400 km, with (ii) aerial sampling (40-250 meters above ground) of mosquitoes using traps tethered to helium filled balloons. We continue to work on identification of the mosquitoes and many other insects that are caught in these high-altitude traps; to date this analysis has established the first report of wind-assisted long-distance migration of malaria vectors. These findings have important implications for malaria control and for the understanding and modeling of malaria transmission. Impact of Iron Availability on Sickling Kinetics in Malian Children To address the Impact of iron availability on sickling kinetics in Malian Children, our specific aims are to: Assess alpha globin gene copy number and iron bioavailability; examine the distribution of intracellular hemoglobin concentration (mean corpuscular hemoglobin concentration, MCHC) and related RBC parameters; measure hemoglobin composition and oxygen affinity: quantify sickling kinetics of fresh RBCs ex vivo using an automated microscope; and determine rates of hemolysis and red cell turnover in vivo. Together, these measurements will provide the first comprehensive evaluation of naturally existing heterogeneity of hemoglobin composition in the red blood cell and its impact on the kinetics of sickling. Malaria Transmission Transmission of malaria is a critical aspect of the parasite life cycle but is poorly understood, and we have increased our efforts to investigate parasite sexual stages, malaria transmission in the field, and the biology of the mosquito vector. In May of 2014 we completed a clinical protocol studying 500 individuals of all ages in Kenieroba (13-I-N107). Examining more than 10,000 samples adn using sensitive molecular methods rather than slide reading, we found that a relatively high proportion of individuals of all ages carry malaria parasites: prevalence was highest in November of 2013 during the wet season and lowest in May of 2014. Unlike most studies, we assessed longitudinal prevalence of parasites in the population and by a linear regression model, only age and gender showed significant effects on the longitudinal prevalence (p<0.0001 and p=0.0008, respectively), while other host factors did not. We showed for the first time that increasing P. falciparum longitudinal prevalence throughout the year was associated with decreasing risk of clinical malaria. This suggests that those with persistent parasite carriage acquire stronger protective immunity against clinical malaria. Using the parasite DNA barcoding technique and have found that most individuals in Kenieroba carry more than one clone of P. falciparum and that this distribution does not change from the wet season to the dry season. Further, using RT-PCR to test for gametocyte mRNA encoding Pfs25, we have determined that 50-80% of people that harbor parasite DNA also have detectable gametocytes throughout the year, showing that no one group can be uniquely targeted for interventions to reduce transmission. Screening of Malian Cohort for Antibodies to Asexual Stage Antigens We have also used the samples derived from the Malian cohort to study the responses of individuals to one of the major blood stage vaccine candidates PfRh5 and other proteins in its complex. This complex is critical for invasion of host red blood cells and promising clinical trial results have recently become available (S. Draper et al.). We have quantitated antibodies to PfRH5 and other complex proteins in Malians of various ages and established that the concentrations of anti-PfRh5 antibodies are far lower (100x less) than has been seen in a vaccine study in nave volunteers from the UK. The basis for this is not clear given the extensive exposure of the Malians but this is important information as a prelude to clinical trials in the field with members of this complex. Naturally Occurring Wolbachia in Anopheline Mosquitoes from Mali Wolbachia is a bacterium commonly found in arthropods that is known to reduce susceptibility of culicine mosquitoes to several Arboviral infections. Anopheline mosquitoes were thought to be naturally refractory to Wolbachia infection. However, we identified a naturally-occurring strain of Wolbachia in An. gambiae s.l. mosquitoes in the Malian villages of Kenieroba and Dangassa, different from strains previously identified in Burkina Faso. Field and laboratory data indicate that Wolbachia infection reduces the prevalence and intensity of sporozoite infection. We carried out experimental P. falciparum infections and found that Wolbachia does not affect the early stages of Plasmodium in the mosquito, but the prevalence and intensity of sporozoite infection was significantly reduced in females infected with Wolbachia (see PNAD publication). More recently we have trained a Malian investigator to do genomic DNA extractions from field-caught desiccated mosquitoes, and we have optimized a new single-PCR detection assay to detect Wolbachia. We will examine the seasonal variation in prevalence of infection in Kenieroba and Dangassa and survey other villages in the area to establish the range of distribution of Wolbachia in this region, as well as different larval habitats, to establish whether Wolbachia infection is widespread or limited to specific larval breeding sites. Leishmania in Mali Leishmania is another parasitic disease endemic to Mali. We have examined the impact of malaria control measures such as long-lived be nets and indoor residual spraying, showing that these interventions also impact sandflies and Leishmania infection. We have also done a study of the co-endemicity of filarial and Leishmania infections in different settings in Mali. The presence of the two infections transmitted by mosquitoes and sandflies makes integrated vector control very important in areas like these.